1. Field of the Invention
The present invention relates to a method for the production and purification of leukocyte interferon. More particularly, the present invention is directed to an improved method for producing and purifying human interferon derived from human leukocyte or lymphoblastoid cell origin.
2. Description of the Prior Art
Interferons are proteinaceous substances which are capable of making living cells resistant to infection caused by viral and nonviral agents. Interferons are also known to act as potent immuno-regulators and as antineoplastic agents.
Interferon activity derived from leukocytes is due to the presence of at least 9-11 different protein components. Therefore, it is important that the isolation of interferon from biological fluids should preserve all existing components. Moreover, the purification procedure used to isolate interferon in its natural multi-component state should be carried out under mild enough conditions to prevent any structural or conformational modifications of the underlying individual proteins. In order for the full prophylactic and therapeutic potential of interferons to be realized, it is essential that the conditions described above be fully met.
Several procedures have been developed for the purification of human interferon derived from leukocytes and lymphoblastoid cells (Namalva). These know procedures usually involve several complex steps and are not ideally suited for efficient large scale, commmercial production. Also, the recovery of interferon produced by these procedures varies considerably. The final interferon yield may be anywhere between 10% to 70% of the starting material.
The majority of human interferon used today is produced and purified essentially according to the procedure developed by Kari Cantell et al. as described in "Partial Purification Of Human Leukocyte Interferon On A Large Scale" appearing in Methods In Enzymology, Volume 78, pages 499-505 (and the other articles cited therein). These methodologies involve the use of ammonium chloride to lyse red blood cells, low pH treatment of the crude interferon preparation for the purpose of inactivating the inducer virus and the use of harsh chemicals such as ethanol or potassium thiocyanate. Others have purified leukocyte interferon by using various sorbents including glass or a specific antibody immobilized to a solid support. In either case, interferon desorption was accomplished by using harsh chemicals like ethanol, potassium thiocyanate or low ph (highly acidic) buffers. However, the human interferon purified by these procedures may not be in its natural form. The use of the ammonium chloride to lyse red blood cells has been shown to be responsible for the loss of about 30% to 40% of leukocytes. Ammonium chloride treatment also damages granulocytic membranes causing the release of proteases capable of inactivating interferon. The low pH treatment results in the loss of the "pH2-labile" leukocyte interferon component which is believed to have a significantly higher level of antitumor activity than the "pH-stable" interferon component. The use of harsh chemicals such as potassium thiocyanate which is a chaotropic salt, is known to disrupt the tertiary structure of proteins, thereby causing a significant loss of antiviral activity and a considerable variation in the amounts of interferon recovered from one batch to another.
In addition, conventional interferon purification technology usually involves several steps requiring numerous intervening concentration and dialysis operations. Such procedures greatly increase the time required for interferon purification and often lead to substantial product loss due to the frequency of mechanical handling required.
The present invention achieves results never before attained in the art, namely efficient, economical commercial production of human leukocyte interferon in its natural form. Both the preparation of cells and the composition of interferon induction media are substantially new. The purification procedure, according to the present invention, is milder than any other such procedures known to date. Finally, the new process, as described herein, successfully avoids all of the drawbacks associated with the Cantell and other methodologies recited above. In particular, this process specifically results in the preservation of the "ph2-labile" interferon component which, until now, has been destroyed by all other state of the art procedures.